Thompson Samuel, Zhang Yanrong, Yang Zijian, Nichols Lisa, Fordyce Polly M
Department of Genetics, Stanford University, Stanford, CA 94305, USA.
Stanford Shared FACS Facility, Stanford University, Stanford, CA 94305, USA.
Adv Mater Interfaces. 2025 Feb 3;12(3). doi: 10.1002/admi.202400403. Epub 2024 Dec 4.
Biphasic environments can enable successful chemical reactions where any single solvent results in poor substrate solubility or poor catalyst reactivity. For screening biphasic reactions at high throughput, a platform based on microfluidic double emulsions can use widely available FACS (Fluorescence Activated Cell Sorting) machines to screen millions of picoliter reactors in a few hours. However, encapsulating biphasic reactions within double emulsions to form FACS-sortable droplet picoreactors requires optimized solvent phases and surfactants to produce triple emulsion droplets that are stable over multi-hour assays and compatible with desired reaction conditions. This work demonstrates such FACS-sortable triple emulsion picoreactors with a fluorocarbon shell and biphasic octanol-in-water core. First, surfactants are screened to stabilize octanol-in-water emulsions for the picoreactor core. With these optimized conditions, stable triple emulsion picoreactors (>70% of droplets survived to 24 hr), produced protein in the biphasic core via cell-free protein synthesis are generated, and sorted these triple emulsions based on fluorescence using a commercial FACS sorter at >100 Hz with 75-80% of droplets recovered. Finally, an in-droplet lipase assay with a fluorogenic resorufin substrate that partitions into octanol is demonstrated. These triple emulsion picoreactors have the potential for future screening bead-encoded catalyst libraries, including enzymes such as lipases for biofuel production.
双相环境能够促成成功的化学反应，而任何单一溶剂都会导致底物溶解度不佳或催化剂反应活性低下。为了高通量筛选双相反应，基于微流控双重乳液的平台可以利用广泛使用的荧光激活细胞分选仪（FACS），在数小时内筛选数百万个皮升反应器。然而，将双相反应封装在双重乳液中以形成可通过FACS分选的液滴皮升反应器，需要优化溶剂相和表面活性剂，以产生在数小时的测定过程中稳定且与所需反应条件兼容的三重乳液液滴。这项工作展示了具有氟碳外壳和水包正辛醇双相内核的可通过FACS分选的三重乳液皮升反应器。首先，筛选表面活性剂以稳定用于皮升反应器内核的水包正辛醇乳液。在这些优化条件下，生成了稳定的三重乳液皮升反应器（>70%的液滴存活至24小时），通过无细胞蛋白质合成在双相内核中产生蛋白质，并使用商用FACS分选仪以>100 Hz的频率基于荧光对这些三重乳液进行分选，回收的液滴比例为75 - 80%。最后，展示了一种在液滴中进行的脂肪酶测定，使用一种可分配到正辛醇中的荧光素底物试卤灵。这些三重乳液皮升反应器有潜力用于未来筛选珠子编码的催化剂文库，包括用于生物燃料生产的脂肪酶等酶。
